Given the importance of HIV-1 in human disease and the wealth of information about the structure and function of the HIV-1 proteins, including reverse transcriptase (RT), we want to understand HIV-1 replication in more detail. We are particularly interested in correlating the structure of HIV-1 RT, and the biochemical properties of wild-type and mutant RTs, with the process of reverse transcription in cells infected with a one-round HIV-1 vector. In the experiments with HIV-1 vectors, we can monitor several of the important steps in reverse transcription and the specificity of RNase H cleavage. Fortunately, many of the lessons we have learned in developing the RCAS vectors are useful in planning the HIV-1 vector experiments, and some of the tools we have developed (in particular, the shuttle cassette from the RCAS-derived RSVP vectors) can, with some modifications, be used in the HIV-1 system. We believe that the interactions between HIV-1 RT and the different types of nucleic acid substrates it uses are important for the ability of the enzyme to constrain, bend, and properly position its nucleic acid substrates and that this positioning is critical for both the RNase H and polymerase activities. As a consequence, we are particularly interested in mutating amino acids that contact the nucleic acid substrates to determine their roles in reverse transcription. We plan to select pseudorevertants (compensatory mutations) for some of the mutations that have interesting in vivo phenotypes. We are also interested in determining why the process of reverse transcription is significantly more efficient in an infected cell than it is in an in vitro system comprised of purified components. We will try to develop methods to identify and isolate reverse transcription complexes (RTCs) and preintegration complexes (PICs) from infected cells with the goal of identifying the viral and host proteins present in RTCs and PICs. We have also developed HIV-1 vectors that carry many of the common mutations associated with resistance to nucleoside RT inibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). These vectors are being used to evaluate candidate compounds;the long-term goal of our work with HIV-1 RT and the HIV-1 vectors is the development of more effective drugs. The work intended to discover new NRTIs is being done in collaboration with Dr. Victor Marquez (Laboratory of Medicinal Chemistry);the work developing new NNRTIs is being done in collaboration with Dr. Craig Thomas (NIH Chemical Genomics Center), Dr. Gary Pauly (Laboratory of Medicinal Chemistry), and Dr. Eddy Arnold. We are using the HIV-1 vectors to help Dr. Terrence Burke (Laboratory of Medicinal Chemistry, NCI) and Dr. Yves Pommier (Laboratory of Molecular Pharmacology, NCI) evaluate the effectiveness of compounds that target HIV-1 integrase. We are continuing to use the RCAS retroviral vector system to study viral replication. These vectors are replication competent, grow to high titer, and are safe and easy to use, both in cultured cells and in animals. Although we have worked with the RCAS vectors both in chickens and in mice for many years, we have made some difficult decisions about which experiments to pursue, and have eliminated all of the work with the RCAS vectors in chickens and in chicken embryos. We are continuing to do mouse experiments with the RCAS vectors, but have reduced the scope of the work, focusing on determining the ability of the RCAS vectors to sustain the expression of a foreign gene in experiments involving the ex vivo treatment of hematopoietic stem cells which are engrafted into mice. We are doing control infection/engraftment experiments in mice with murine leukemia virus (MLV) and HIV-1 vectors that carry the same promoters and marker genes. We have developed an RCAS shuttle vector and have used it to show that linear viral DNAs with one defective end can be inserted into the host genome efficiently, although the defective end does not appear to be inserted by integrase. [Corresponds to Hughes Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program]